Novel radioactive estratrienes

ABSTRACT

Novel radioactive Δ 4 ,9,11 -estratrienes marked with iodine 125 or 131 having the formula ##STR1## wherein R is selected from the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms and acyl of an organic carboxylic acid of 2 to 12 carbon atoms, R 1  is the reminder of an amino acid of the formula R 1  NH 2  or a derivative thereof being marked with iodine 125 or 131 and the wavy lines indicate the --OR is in the α- or β-position and the oximido group is in the syn or anti position, a process and intermediates for their preparation, antigens prepared therefrom and use thereof to prepare antibodies.

STATE OF THE ART

U.S. Pat. No. 4,339,390 describes radiolabeling of cortisol-21-acetateand British Pat. No. 1,604,864 relates to iodinating progesteronederivatives. French Pat. No. 2,397,426 radioactive steroids and FrenchPat. No. 2,250,745 describes methyltestosterone-3-(0-carboxymethyl)oximino-tyrosinate and its preparation.

OBJECTS OF THE INVENTION

It is an object of the invention to provide the radioactive iodinemarked steroids of formula I and a process and novel intermediates fortheir preparation.

It is a further object of the invention to provide antigens of saidradioactive iodine marked steroids of formula I and bovine seric albuminand human seric albumin.

It is an additional object of the invention to provide novel antibodiesand their use.

These and other objects and advantages of the invention will becomeobvious from the following detailed description.

THE INVENTION

The novel radioactive Δ⁴,9,11 -estratrienes marked with iodine 125 or131 of the invention have the formula ##STR2## wherein R is selectedfrom the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms andacyl of an organic carboxylic acid of 2 to 12 carbon atoms, R₁ is thereminder of an amino acid of the formula R₁ NH₂ or a derivative thereofbeing marked with iodine 125 or 131 and the wavy lines indicate the --ORis in the α- or β-position and the wavy line attached to the nitrogenindicates the syn or anti position.

The derivatives of the amino carboxylic acids are preferably adecarboxylated derivative or a lower alkyl ester of the amino acid. Thepreferred amino acids marked with radioactive iodine are selected fromthe group consisting of histidine, tyrosine, histamine, tyramine andmethyl tyrosinate marked with ¹²⁵ I or ¹³¹ I.

Among the preferred compounds of formula I are the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol coupled with ¹²⁵ Ihistamine of the formula ##STR3## with the iodine in the 2- or5-position; the anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol coupled with ¹²⁵ I histamine of the formula ##STR4##with the iodine in the 2- or 5-position; the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol coupled with ¹²⁵ Ihistamine of the formula ##STR5## with the iodine in the 2- or5-position; the anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol coupled with ¹²⁵ I histamine of the formula ##STR6##with the iodine in the 2- or 5-position; a mixture of the syn and antiisomers of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β- or 17α-olcoupled with ¹²⁵ I histamine with the iodine in the 2- or 5-position.

The novel process of the invention for the preparation of the compoundsof formula I comprises reacting a compound of the formula ##STR7##wherein R and the wavy line have the above definitions with acarboxymethoxyamine halide in the presence of a base to obtain acompound of the formula ##STR8## wherein the wavy line in the17-position has the above definition and the wavy line attached to thenitrogen is a mixture of anti and syn isomers and R₂ is hydrogen andthen either fixing the acid function of the product with an activatorgroup of a carbonyl function to obtain a compound of the formula##STR9## wherein R₂ is an activating group of the carbonyl function andthe wavy line on the nitrogen indicates a mixture of syn and antiisomers and reacting the latter with an amino acid marked with ¹²⁵ I or¹³¹ I or a derivative thereof to obtain the corresponding compound offormula I as a mixture of the syn and anti isomers which can beoptionally separated or reacting a compound of formula III_(A) as amixture of syn and anti isomers with an esterification agent of analkanol of 1 to 6 carbon atoms to obtain a mixture of the syn and antiisomers of a compound of the formula ##STR10## wherein R₂ is alkyl of 1to 6 carbon atoms, separating the isomers, individually saponifying thesaid esters to obtain the compound of formula III_(A) in the form of itsindividual anti and syn isomers wherein R₂ is hydrogen, fixing the acidgroup of the said isomers with a carbonyl activator to obtain theindividual syn and anti isomers of the compound of formula III_(C)wherein R₂ is the carbonyl activator function and reacting the latterwith an amino acid marked with ¹²⁵ I or ¹³¹ I or a derivative thereof toobtain the individual syn and anti isomers of formula I.

In a preferred process of the invention, the amino acid marked withiodine or a derivative thereof is selected from the group consisting ofhistidine, tyrosine, histamine, tyramine and methyl tyrosinate and thereaction of the compound of formula II is effected withcarboxymethoxyamine hemihydrochloride in the dark under an inertatmosphere in the presence of sodium hydroxide. The fixing of the acidfunction with a carbonyl activator is preferably effected with an alkylhaloformate in the presence of a tertiary base under an inert atmosphereand anhydrous conditions which results in the formation of a group ofthe formula ##STR11## wherein AlK is alkyl of 1 to 6 carbon atoms.

More preferably, the alkyl haloformate is isobutyl chloroformate and thereaction is effected in the presence of tri-n-butylamine. Theesterification agent is diazomethane and the saponification of the estergroup is effected with methanolic sodium hydroxide. The reaction of thecompound of formula III_(C) wherein R₂ is a carbonyl activating group iseffected with histamine marked with ¹²⁵ I or ¹³¹ I in the dark under aninert atmosphere. The activation of the carbonyl group of formulaIII_(A) where R₂ is hydrogen is effected by N-hydroxy-succinimide ordicyclohexylcarbodiimide to generate the activated group of thecarbonyl.

The more preferred embodiment of the process of the invention comprisesreacting Δ⁴,9,11 -estratriene-17β-ol-3-one or Δ⁴,9,11-estratriene-17α-ol-3-one to obtain a mixture of the corresponding synand anti isomers or the individual syn or anti isomers of the formulae##STR12## with iodine in the 2- or 5-position.

The compounds of formula I are useful for radioimmunological dosages ofΔ⁴,9,11 -estratriene-17α- and 17β-ols in bile, urine, feces, plasma andanimal and human tissues and in foods of humans and warm-bloodedanimals.

The novel intermediates of the invention for the preparation of thecompounds of formula I are the compounds in the form of syn and antiisomers and mixtures thereof of the formula ##STR13## wherein R ishydrogen, alkyl of 1 to 6 carbon atoms or acyl of an organic carboxylicacid of 2 to 12 carbon atoms, R₂ is hydrogen, alkyl of 1 to 6 carbonatoms or a carbonyl activating group, the wavy line in the 17-positionindicates the α- or β-position and the wavy line attached to thenitrogen indicates the syn or anti position. The carbonyl activatorgroup is preferably --COOAlK with AlK being alkyl of 1 to 6 carbonatoms.

Among the preferred compounds of formula III are the syn and antiisomers and mixtures thereof of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol or 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol or 3-carbethoxymethyloximino-Δ⁴,9,11-estratriene-17β-ol or 3-carbethoxymethyloximino-Δ⁴,9,11-estratriene-17α-ol, the mixed anhydride of isobutyl formate and3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol in the form of itsanti and syn isomers and mixtures thereof of the formula ##STR14##wherein the wavy line has the above definition and the mixed anhydrideof isobutyl formate and 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol in the form of its anti and syn isomers and mixturesthereof of the formula ##STR15##

The compounds of formula III, especially those wherein R₂ is hydrogen,are useful starting compounds for the preparation of antigens, equallynecessary for radioimmunological doses of Δ⁴,9,11 -estratriene-17β- and17α-ol-3-ones. The use of the compounds of formula III conjugated withbovine seric albumin (BSA) or human seric albumin (HSA) to obtain anantigen is also part of the invention.

In a preferred process of the invention for the preparation of antigenswith the compounds of formula III in which R₂ is hydrogen comprisesreacting a compound of formula III_(A) in the form of its anti isomer orsyn isomer or mixtures thereof when R₂ is hydrogen and R has the abovedefinition after activation of the carbonyl function with an alkylhaloformate in the presence of a tertiary base under anhydrousconditions and an inert atmosphere to obtain a mixed anhydride offormula III^(C) wherein R₂ is --COOAlK and AlK is alkyl of 1 to 6 carbonatoms and conjugating the latter with bovine seric albumin (BSA) orhuman seric albumin (HSA) to obtain the desired antigen.

In the preferred mode of the latter process, the compound of formula IIIis reacted with isobutyl chloroformate in anhydrous dioxane in thepresence of tri-n-butylamine under an inert atmosphere and the mixedanhydride is reacted with a solution of bovine seric albumin or humanseric albumin in a mixture of water and dioxane under an inertatmosphere.

Examples of preferred antigens starting from the syn or anti isomer ormixture thereof of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ORare antigens of the formula ##STR16## wherein n is 20 to 30 and startingfrom 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-OR in the form ofits anti or syn isomer or mixtures thereof are antigens of the formula##STR17## wherein n is 20 to 30. The antigens of formulae IV, V, VI andVII are a part of the invention.

The novel method of the invention for the preparation of antibodiescomprises administering to warm-blooded animals an antigen of theinvention in the presence of an adjuvant to obtain serum containingantibodies against Δ⁴,9,11 -estratriene-17β-ol-3-one and Δ⁴,9,11-estratriene-17α-ol.

The compounds of formula I and especially the syn and anti isomers andmixtures thereof of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol¹²⁵ I histamine or 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol¹²⁵ I histamine are used in radioimmuniological dosage of Δ⁴,9,11-estratriene-17α- and 17β-ol-3-ones and permits dosages of theseproducts in biological liquids and tissues and in human and animalalimentation. The radioimmunological dosage is effected by the method ofBergson et al [Hormone, Vol. 4 (1964), p. 557] and Abraham [Journal ofChemical Endocrinonal metab, Vol. 29 (1969), p. 866].

In the following examples there are described several preferredembodiments to illustrate the invention. However, it is to be understoodthat the invention is not intended to be limited to the specificembodiments.

EXAMPLE 1 Syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β- and 17α-ol

STEP A: Syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol

3.05 g of carboxymethoxyamine hemihydrochloride and 15 ml of N sodiumhydroxide solution were successively added under an inert atmosphere inthe dark to a suspension of 3.29 g of Δ⁴,9,11 -estratriene-17β-ol-3-onein 85 ml of ethanol and the mixture was stirred in the dark at roomtemperature for about 3 hours and was poured into iced water containinghydrochloric acid. The mixture was vacuum filtered and the product waswashed with water until the wash water was neutral and dried underreduced pressure to obtain 3.86 g of product. The latter waschromatographed over silica gel and was eluted with a 90-10-1 methylenechloride-methanol-acetic acid mixture to obtain a mixture of syn andanti isomers of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol withan RF=0.35.

NMR Spectrum (90 MHz):

529 Hz (4-hydrogen of anti isomer); 558-584 Hz (4-hydrogen of masked synisomer).

STEP B: Syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol

Using the procedure of Step A, a mixture of 1.59 g of Δ⁴,9,11-estratriene-17α-ol-3-one, 40 ml of ethanol, 1.5 g ofcarboxymethoxyamine hemihydrochloride and 15 ml of N sodium hydroxidesolution were reacted to obtain 1.75 g of product. The latter waschromatographed over silica gel and was eluted with a 7-3cyclohexane-ethyl acetate mixture to obtain a mixture of the syn andanti isomers of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol withan Rf=0.25.

EXAMPLE 2 Separation of syn and anti isomers of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol

STEP A: Syn and anti isomers of 3-carbethoxymethyloximino-Δ⁴,9,11-estratriene-17β-ol

25 ml of diazomethane were added with stirring at 0° C. under an inertatmosphere to a suspension of 1.3 g of a mixture of syn and anti isomersof 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol in 20 ml ofmethylene chloride and the mixture was stirred for one hour at 0° C. Thedecanted organic phase was evaporated to dryness under reduced pressureto obtain 1.53 g of an oil product which was dissolved in 3 ml of ethylacetate. The solution was chromatographed over silica gel and elutionwith a 1-1 cyclohexane-ethyl acetate mixture yielded 750 mg of the antiisomer of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol in the formof an ester with an Rf=0.32 and a second oil fraction of 350 mg of thesyn isomer with an Rf=0.28. The separated isomers were saponified andanalyzed in the acid form to confirm the syn and anti structures.

STEP B: Syn isomer of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol

A mixture of 350 mg of the syn isomer of Step A, 5 ml of methanol and 1ml of sodium hydroxide was stirred in the dark under an inert atmosphereat room temperature for one hour and the mixture was cooled to 0° C. andadmixed with 1 ml of concentrated hydrochloric acid. The mixture wasvacuum filtered and the product was dried under reduced pressure toobtain 295 mg of the syn isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol.

NMR Spectrum (90 MHz):

570-590 Hz (4,-11- and 12-hydrogens of syn isomer)

UV Spectrum (ethanol):

    ______________________________________                                        Inflex. towards                                                                           313 nm    E.sub.1.sup.1 = 883                                     Max. at     325 nm    E.sub.1.sup.1 = 1144                                                                      ε = 39,300                          Inflex. towards                                                                           335 nm    E.sub.1.sup.1 = 1098                                    ______________________________________                                    

Circular dichroism (ethanol):

    ______________________________________                                        Max. at       231 nm      Δε = +2.75                            Inflex. towards                                                                             255 nm      Δε = -2.25                            Inflex. towards                                                                             290 nm      Δε = -2.7                             Max. at       302 nm      Δε = -3.8                             Max. at       315 nm      Δε = -4.5                             Max. at       331 nm      Δε =  2.5                             Max. at       347 nm      Δε = +1.3                             ______________________________________                                    

STEP C: Anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol

Using the procedure of Step B, the 750 mg of the anti isomer of Step A,10 ml of methanol and 1.6 ml of sodium hydroxide were reacted to obtain660 mg of the anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol.

NMR Spectrum (90 MHz):

526 Hz (4-hydrogen of anti isomer).

UV Spectrum (ethanol):

    ______________________________________                                        Inflex. towards                                                                           310 mn    E.sub.1.sup.1 = 855                                     Inflex. towards                                                                           335 nm    E.sub.1.sup.1 = 1010                                                                      ε = 39,300                          Max. at     322 nm    E.sub.1.sup.1 = 1144                                    ______________________________________                                    

Circular dichroism (ethanol):

    ______________________________________                                        Max at        231 nm      Δε = +3.0                             Max. at       253 nm      Δε = -1.05                            Inflex. towards                                                                             290 nm      Δε = -2.7                             Max. at       300 nm      Δε = -4.2                             Max. at       315 nm      Δε = -4.6                             Max. at       329 nm      Δε = -1.6                             Max. at       343 nm      Δε = +2.75                            ______________________________________                                    

EXAMPLE 3 Syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol

STEP A: 3-carbethoxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol

Using the procedure of Step A of Example 2, 1.64 g of a mixture of thesyn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol of Example 1, 26 ml of methylene chloride and 35 mlof diazomethane were reacted to obtain 1.55 g of a mixture of syn andanti of 3-carboxymethyloximio-Δ⁴,9,11 -estratriene-17α-ol. The mixturewas chromatographed over silica gel and was eluted with a 7-3cyclohexaneethyl acetate mixture obtain 580 mg of the presumed antiisomer with an Rf=0.17 and a second fraction of 490 mg of the presumedsyn isomer with an Rf=0.13. The products were saponified and analyzed inthe acid form.

STEP B: Anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α-ol

Using the procedure of Step B of Example 2, 580 mg of the anti isomer ofStep A, 7 ml of methanol and 1.3 ml of sodium hydroxide solution werereacted to obtain 440 mg of anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol.

NMR Spectrum (90 MHz):

532 Hz (4-hydrogen of anti isomer)

UV Spectrum (1-4 ethanol-water):

    ______________________________________                                        Inflex. towards                                                                           312 nm    E.sub.1.sup.1 = 874                                     Max. at     323 nm    E.sub.1.sup.1 = 1132                                                                      ε = 39,000                          Inflex. towards                                                                           335 nm    E.sub.1.sup.1 = 1010                                    ______________________________________                                    

Circular Dichroism (1-4 ethanol-water):

    ______________________________________                                        Max. at 251 nm       Δε = -2.4                                  Max. at 303 nm       Δε = -2.9                                  Max. at 315 nm       Δε = -2.8                                  Max. at 349 nm       Δε = +2.8                                  ______________________________________                                    

STEP C: Syn isomer of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol

Using the procedure of Step C of Example 2, 490 mg of the presumed synisomer of Step A, 6,5 ml of methanol and 1,1 ml of sodium hydroxidesolution were reacted to obtain 325 mg of syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estradiene-17α-ol.

NMR Spectrum (90 MHz):

532 Hz for H₂

585 Hz for H₄ of syn isomer

UV Spectrum (1-4 ethanol-water)

    ______________________________________                                        infl.      312 nm        E.sub.1.sup.1 = 888                                  max.       325 nm        E.sub.1.sup.1 = 1168                                 infl.      335 nm        E.sub.1.sup.1 = 1136                                 ______________________________________                                    

Circular dichroism (1-4 ethanol-water)

    ______________________________________                                        max. 250 nm          Δε = -3.3                                  max. 303 nm          Δε = -3.0                                  max. 316 nm          Δε = -3.1                                  max. 350 nm          Δε = +2.6                                  ______________________________________                                    

EXAMPLE 4 Antigens of seric bovine albumin (BSA) and anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol

0.23 ml of tri-n-butylamine were added with stirring under an inertatmosphere to a solution of 170 mg of the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol in 5 ml of dioxaneand after reducing the temperature towards 13° C., 0.65 ml of isobutylchloroformate were added thereto. The mixture was stirred at 13° C. for30 minutes to obtain a solution of the mixed anhydride of isobutylchloroformate and 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol.

2 ml of dioxane were added with stirring under an inert atmosphere to asolution of 690 mg of BSA in 20 ml of iced water and the solution wasstirred for 20 minutes. Then 20 ml of dioxane and then 0.7 ml of Nsodium hydroxide solution were successively added to the mixture and themixed anhydride solution was added to the BSA solution. The mixture wasstirred at 4° C. for four hours and then 50 ml of distilled water wereadded thereto. The pH of the mixture was adjusted to 4.1 by addition ofN hydrochloric acid. The mixture was filtered and the solid product wastaken up in 50 ml of aqueous 1% sodium bicarbonate solution. Theresulting solution was subjected to ultra filtrations and then passed505 ml of water to extract the compounds with a molecular weight <10 to15,000. The volume was adjusted to 23 ml and the solution was subjectedto lyophilization for 24 hours to obtain 759 mg of antigens of sericbovine albumin (BSA) and anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol having 24 to 25 steroid groups per mole.

UV Spectrum (water in presence of dioxane):

    ______________________________________                                        Max. at 325 nm         E.sub.1.sup.1 = 125                                    Inflex. towards 339 nm E.sub.1.sup.1 = 98                                     ______________________________________                                    

Circular Dichroism (water in presence of dioxane):

    ______________________________________                                        Max. at 209 nm     Δ E.sub.1.sup.1 = -0.350                             Max. at 220 nm     Δ E.sub.1.sup.1 = -0.305                             Max. at 335 nm     Δ E.sub.1.sup.1 = -0.020                             ______________________________________                                    

EXAMPLE 5 Antigen of BSA and the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol

Using the procedure of Example 4, 170 mg of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol and 690 mg of BSAwere reacted to obtain 912 mg of antigen of BSA and the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol having 22 to 23steroid groups per mole.

UV Spectrum (water in presence of dioxane):

    ______________________________________                                        Max. at 325 nm         E.sub.1.sup.1 = 111                                    Inflex. towards 340 nm E.sub.1.sup.1 = 89                                     ______________________________________                                    

Circular dichroism:

    ______________________________________                                        Max. at 213 nm     Δ E.sub.1.sup.1 = -0.328                             Max. at 223 nm     Δ E.sub.1.sup.1 = -0.324                             Max. at 305 nm     Δ E.sub.1.sup.1 = -0.010                             Max. at 316 nm     Δ E.sub.1.sup.1 = -0.009                             Max. at 351 nm     Δ E.sub.1.sup.1 = +0.011                             ______________________________________                                    

EXAMPLE 6 Antigen of BSA and syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol

Using the procedure of Example 4, 170 mg of the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol and 690 mg of BSAwere reacted to obtain 902 mg of the antigen of BSA and syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol containing 22 to 24steroid groups per mole.

UV Spectrum (water in presence of dioxane):

    ______________________________________                                        Max. at 325 nm         E.sub.1.sup.1 = 118                                    Inflex. towards 340 nm E.sub.1.sup.1 = 100                                    ______________________________________                                    

Circular dichroism:

    ______________________________________                                        Max. at 210 nm     Δ E.sub.1.sup.1 = -0.396                             Max. at 222 nm     Δ E.sub.1.sup.1 = -0.010                             Max. at 317 nm     Δ E.sub.1.sup.1 = -0.009                             Max. at 351 nm     Δ E.sub.1.sup.1 = +0.012                             ______________________________________                                    

EXAMPLE 7 Antigen of BSA and syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol

Using the procedure of Example 4, BSA and the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol were reacted to formthe antigen of BSA and syn isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol.

EXAMPLE 8

Using the procedure of Example 4 an antigen was prepared from a mixtureof the syn and anti isomers of 3-carboxymethyloximino Δ⁴,9,11-estratriene-17β and 17β-ols and BSA.

EXAMPLE 9

Using the procedure of Example 4, an antigen was prepared from a mixtureof the syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17α- and 17β-ols and human seric albumin (HSA) as well asthe antigens of HSA and the individual syn and anti isomers thereof.

EXAMPLE 10 Couple of (I¹²⁵) histamine and 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol

10 ml of 1-5 tri-n-butylamine-tetrahydrofuran mixture and 10 ml of 1-10isobutyl chloroformate-tetrahydrofuran mixture were added with coolingunder an inert atmosphere with stirring to a solution of 2.4 mg of amixture of the syn and anti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol in 50 ml of tetrahydrofuran and the mixture wasstirred with cooling for 30 minutes. 3.4 ml of tetrahydrofuran wereadded to the mixture to obtain a solution of the mixed anhydride ofisobutyl chloroformate and the syn and anti isomer mixture of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol which was used infra.

1 mCi of sodium iodide ¹²⁵ I (specific activity of 2000 Ci mmol) andthen 50 μg of chloroamine T in 10 μl of distilled water weresuccessively added to 10 μl of a solution of 2 mM of histamine in abuffered solution of 0.5M sodium phosphate with a pHof 8 and the mixturewas stirred for about 90 seconds. A solution of 300 μg of sodiummetabisulfate in 10 μl of distilled water were added to the mixture toobtain a solution of (¹²⁵ I) histamine with an Rf=0.1 (chromatographyover silica gel-elution with a 98-2 methanoltriethylamine mixture) whichwas used infra.

50 μl of the mixed anhydride solution were added to the ¹²⁵ I histaminesolution and the mixture was stirred in the dark at about 4° C. for onehour. The mixture was diluted with 0.4 ml of 0.1M aqueous sodiumbicarbonate-solution and was extracted with 1.5 ml of methylenechloride. The organic phase was evaporated to dryness and the residuewas subjected to high performance liquid chromatography. Elution with a97-3 chloroform-methanol mixture yielded a first peak of the couple of¹²⁵ I histamine and the anti isomer of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol with a retention time 38 minutes and a second peakof the couple of ¹²⁵ I histamine and the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol with a retention timeof 42 minutes. The couple of the anti isomer had a total activity of 150μCi and the couple of the syn isomer had a total activity of 50 μCi.

EXAMPLE 11 Couple of ¹²⁵ I histamine and 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol

Using the procedure of Example 9, 2.4 mg of a mixture of the syn andanti isomers of 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-estratriene-17β-ol, 50 ml of tetrahydrofuran and 10 mlof 1-10 isobutyl chloroformate-tetrahydrofuran mixture were reacted toform a solution of the mixed anhydride and 50 μl of the solution werereacted with a ¹²⁵ I histamine solution of Example 9 to obtain a coupleof the ¹²⁵ I histamine and the anti and syn isomers. The residue wassubjected to high performance liquid chromatography to obtain a firstpeak of the couple of ¹²⁵ I histamine and the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol with a retention timeof 34 minutes and a second peak of the couple of ¹²⁵ I histamine and thesyn isomer with a retention time of 42 minutes. The couple of the antiisomer had a total activity of 50 μCi and the couple of the syn isomerhad a total activity of 150 μCi.

Various modifications of the products and processes of the invention maybe made without departing from the spirit or scope thereof and it is tobe understood that the invention is intended to be limited only asdefined in the appended claims.

What we claim is:
 1. A radioactive Δ⁴,9,11 -estratriene marked withiodine 125 or 131 having the formula ##STR18## wherein R is selectedfrom the group consisting of hydrogen, alkyl of 1 to 6 carbon atoms andacyl of an organic carboxylic acid of 2 to 12 carbon atoms, R₁ is thereminder of an amino acid of the formula R₁ NH₂ or a derivative thereofbeing marked with iodine 125 or 131 and the wavy lines indicate the --ORis in the α- or β-position and the oximido group is in the syn or antiposition.
 2. A compound of claim 1 wherein the amino acid marked withiodine ¹²⁵ I or ¹³¹ I is selected from the group consisting ofhistidine, tyrosine, histamine, tyramine and methyl tyrosinate.
 3. Acompound of claim 1 in the form of the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol coupled with ¹²⁵ Ihistamine of the formula ##STR19## wherein the iodine is in the 2- or5-position.
 4. A compound of claim 1 in the form of the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol coupled with ¹²⁵ Ihistamine of the formula ##STR20## wherein the iodine is in the 2- or5-position.
 5. A compound of claim 1 in the form of the syn isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol coupled with ¹²⁵ Ihistamine of the formula ##STR21## wherein the iodine is in the 2- or5-position.
 6. A compound of claim 1 in the form of the anti isomer of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol coupled with ¹²⁵ Ihistamine of the formula ##STR22## wherein the iodine is in the 2- or5-position.
 7. A mixture of claim 1 of the syn and anti isomers of3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α- or 17β-ol coupled with¹²⁵ I histamine with the iodine in the 2- or 5-position.
 8. A processfor the preparation of a compound of claim 1 comprising reacting acompound of the formula ##STR23## wherein R and the wavy line have theabove definitions with a carboxymethoxyamine halide in the presence of abase to obtain a compound of the formula ##STR24## wherein the wavy linein the 17-position has the above definition and the wavy line attachedto the nitrogen is a mixture of anti and syn isomers and R₂ is hydrogenand then either fixing the acid function of the product with anactivator group of a carbonyl function to obtain a compound of theformula ##STR25## wherein R₂ is an activating group of the carbonylfunction and the wavy line on the nitrogen indicates a mixture of synand anti isomers and reacting the latter with an amino acid marked with¹²⁵ I or ¹³¹ I or a derivative thereof to obtain the correspondingcompound of formula I as a mixture of the syn and anti isomers which canbe optionally separated or reacting a compound of formula III_(A) as amixture of syn and anti isomers with an esterification agent of analkanol of 1 to 6 carbon atoms to obtain a mixture of the syn and antiisomers of a compound of the formula ##STR26## wherein R₂ is alkyl of 1to 6 carbon atoms, separating the isomers, individually saponifying thesaid esters to obtain the compoound of formula III_(A) in the form ofits individual anti and syn isomers wherein R₂ is hydrogen, fixing theacid group of the said isomers with a carbonyl activator to obtain theindividual syn and anti isomers of the compound of formula III_(C)wherein R₂ is the carbonyl activator function and reacting the latterwith an amino acid marked with ¹²⁵ I or ¹³¹ I or a derivative thereof toobtain the individual syn and anti isomers of formula I.
 9. The processof claim 8 wherein the amino acid marked with iodine ¹²⁵ I or ¹³¹ I isselected from the group consisting of histidine, tyrosine, histamine,tyramine and methyl tyrosinate.
 10. The process of claim 8 wherein thestarting material is Δ⁴,9,11 -estratriene-17β-ol-3-one or Δ⁴,9,11-estratriene-17α-ol-3-one resulting in a mixture of the correspondingsyn and anti isomers or the individual syn and anti isomers of theformula ##STR27## with iodine in the 2- or 5-position.
 11. A processcomprising administering to warm-blooded animals a radioimmunologicaldose of a compound of claim 1 and determining the amount of Δ⁴,9,11-estratriene-17β- and 17α-ol-3-one in the animal or human biologicalliquids and tissues and alimentation.
 12. A compound in the form of synand anti isomers and mixtures thereof of the formula ##STR28## wherein Ris hydrogen, alkyl of 1 to 6 carbon atoms or acyl of an organiccarboxylic acid of 2 to 12 carbon atoms, R₂ is hydrogen, alkyl of 1 to 6carbon atoms or a carbonyl activating group, the wavy line in the17-position indicates the α- or β-position and the wavy line attached tothe nitrogen indicates the syn or anti position.
 13. A compound of claim12 selected from the group consisting of the syn and anti isomers andmixtures thereof of 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17β-olor 3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol or3-carbethoxymethyloximino-Δ⁴,9,11 -estratriene-17β-ol or3-carbethoxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol, the mixedanhydride of isobutyl formate and 3-carboxymethyloximino-Δ⁴,9,11-estratriene-17β-ol in the form of its anti and syn isomers and mixturesthereof of the formula ##STR29## wherein the wavy lines have the abovedefinitions and the mixed anhydride of isobutyl formate and3-carboxymethyloximino-Δ⁴,9,11 -estratriene-17α-ol in the form of itsanti and syn isomers and mixtures thereof of the formula ##STR30## 14.An antigen comprising a compound of claim 12 wherein R₂ is hydrogenconjugated with human seric albumin or bovine seric albumin.
 15. Aprocess of the preparation of an antigen of claim 14 comprising reactinga compound of claim 12 in the form of its anti isomer or syn isomer ofmixtures thereof wherein R₂ is hydrogen and R has the above definitionafter activation of the carbonyl function with an alkyl haloformate inthe presence of a tetriary base under anhydrous conditions and an inertatmosphere to obtain a mixed anhydride of formula III_(C) wherein R₂ is--COOAlK and AlK is alkyl of 1 to 6 carbon atoms and conjugating thelatter with bovine seric albumin (BSA) or human seric albumin (HSA) toobtain the desired antigen.
 16. An antigen of claim 14 selected from thegroup consisting of syn or anti isomers and mixtures thereof of theformula ##STR31## wherein n is 20 to
 30. 17. A method of preparingantibodies comprising administering to warm-blooded animals an effectiveamount of an antigen of claim 14 in the presence of an adjuvant toobtain serum containing antibodies against Δ⁴,9,11-estratriene-17β-ol-3-one and Δ⁴,9,11 -estratriene-17α-ol.